This application is responsive to NOT-OD-09-058 (NIH Announces the Availability of Recovery Act Funds for Competitive Revision Applications). We seek support from the Recovery Act Funds to expand the scope of the specific aims, research design, and methods of GM065997, which bears the title: Regulation of cullin-RING ligases by Nedd8. Nedd8 is a ubiquitin-like protein that is reversibly conjugated to the cullin subunit of cullin-RING ubiquitin ligases (CRLs). Both the conjugation and deconjugation of Nedd8 are essential to maintain the activity of CRLs in human cells. In the second Aim of the parent application, we proposed to investigate how Nedd8 deconjugation of cullins by the COP9 Signalosome (CSN) is regulated by the recruitment of substrates and other subunits of the ligase complex to the cullin scaffold. In the present application, we propose to expand upon this goal by developing a potent and specific inhibitor of the Nedd8 isopeptidase activity of CSN. We will devise homogeneous fluorescence polarization and fluorescence resonance energy transfer assays to monitor cleavage of Nedd8-Cul1 both in vitro and in vivo, and will employ the in vitro assay to conduct high- throughput screens of chemical libraries. Hits will be subjected to secondary screens designed to evaluate their in vivo efficacy, potency, and selectivity. Our goal is to identify a lead compound with an IC50 of at least 1 ?M in both in vitro and in vivo assays. A lead with these characteristics would be a very useful tool to study the physiological role of CSN-dependent Nedd8 deconjugation in vivo, and to evaluate how this function relates to the substrate specificity of CSN, which is the subject of Aim 2 of the parent application. The experiments proposed here substantially expand the scope of the original peer reviewed Aims of the parent grant by introducing new new methods and approaches that will yield new tools to study the function of CSN. Pursuit of the proposed experiments will accelerate the tempo of research in my lab on the cellular consequences of CRL regulation by Nedd8 deconjugation, and will enable the recruitment of new staff. PUBLIC HEALTH RELEVANCE: Subunits of cullin-RING ubiquitin ligases (CRLs) are mutated or overexpressed in a variety of human diseases including cancer and overexpression of Csn5 drives a gene expression program that is tightly linked to poor prognosis in breast cancer. In addition, the Nedd8 activating enzyme that together with Csn5 mediates the cycle of cullin modification by Nedd8 is the target of a promising anti-cancer drug that is in clinical development. Identification of a chemical that blocks cleavage of Nedd8 from cullins by Csn5 will lead to a deeper understanding of the physiological role of Nedd8 deconjugation and will lead to a better understanding of how overactive Csn5 underlies human disease. Besides being an important research tool, a Csn5 inhibitor has great potential to serve as a starting point for development of new therapeutic drugs to modulate CRL activity for clinical benefit.